Since 2004 technological advances have enabled us to sequence more nucleic acid and generate more data in a shorter amount of time. Decreases in cost per nucleotide sequenced, the initial price of sequencing machines and the complexity of library construction means that whole genome sequencing (WGS) is available in many research labs and an increasing number of public health microbiology labs. I will examine the use of WGS in public health microbiology, particularly the possibility of investigating organisms without culture, the interrogation of genomes where PCR may be unavailable, outbreak investigation, tracking resistance mutations and novel pathogen discovery.

The treatment of endogenous infections caused by commensal Escherichia coli are often complicated by antibiotic resistance. Strains of resistant E. coli in the gastrointestinal tract serve as a reservoir of resistance determinants, and dissemination of resistance genes is often facilitated by conjugative plasmids. It is important to understand these plasmids in order to track the movement of resistance determinants between populations.

Three faecal E. coli isolates from a healthy adult were examined. Two of these (838-98B and -3B) were resistant to ampicillin (Ap), streptomycin (Sm) and sulphamethoxazole (Su). The other (838-50A) was susceptible. 838-50A and -3B were indistinguishable by biochemical and molecular analysis (API20E, phylogenetic group PCR, RAPD). 838-98B was a distinct strain. B/O plasmid replicons were detected in both resistant isolates using PCR-based plasmid replicon typing. A B/O replicon was not detected in the susceptible strain. This suggested that a plasmid bearing a B/O replicon might be responsible for ApSmSu resistance. Conjugation experiments with a laboratory adapted E. coli strain (UB5201) confirmed that the movement of a B/O plasmid from both 838-98B and -3B conferred ApSmSu resistance. Plasmid sequencing revealed that an identical B/O plasmid, p838B-R (94.8kb), was present in 838-98B and -3B, and carried ApSmSu resistance determinants. p838B-R was also observed to mobilise small plasmids, allowing the direction of in situ transfer to be determined.

The observed transfer of antibiotic resistance plasmid p838B-R between two unrelated strains in the gastrointestinal tract highlights the important role commensal bacteria play in the spread of resistance determinants. While not well documented, the association of B/O-type plasmids with antibiotic resistance is evident not only through p838B-R but also other available plasmid sequences. Further studies will allow us to determine the extent to which these plasmids influence antibiotic resistance in commensal E. coli.

Metagenomics is a valuable tool for the study of microbial communities but has been limited by the difficulty of “binning” the resulting sequences into groups corresponding to the individual species and strains that constitute the community. Moreover, there are presently no methods to track the flow of mobile DNA elements such as plasmids through communities or to determine which of these are co-localized within the same cell. We address these limitations by applying Hi-C, a technology originally designed for the study of three-dimensional genome structure in eukaryotes, to measure the cellular co-localization of DNA sequences. We leveraged Hi-C data generated from a synthetic metagenome sample to accurately cluster metagenome assembly contigs into a small number of groups that differentiated the genomes of each species. The Hi-C data also associated plasmids with the chromosomes of their host and with each other orders of magnitude more frequently than to other species. We further demonstrated that Hi-C data is highly informative for resolving strain-specific genes and nucleotide substitutions between two closely related E. coli strains, K12 DH10B and BL21 (DE3), indicating such data may be useful for high-resolution genotyping of microbial populations. Our work demonstrates that Hi-C sequencing data provide valuable information for metagenome analyses that are not currently obtainable by other methods. This application of Hi-C has the potential to provide new perspective in the study of thefine-scale population structure of microbes, how antibiotic resistance plasmids (or other genetic elements) mobilize in microbial communities, and the genetic architecture ofheterogeneous tumor clone populations.

Registration Closed

24-25 February 2014
The Australian Museum

Microbiology is undergoing a revolution bought about by advances in next-generation DNA sequencing technology. Researchers are now required to understand an array of bioinformatics principles and tools to interpret the vast amounts of data being generated. Presented by leading Australian researchers, TOAST is a 2-day event aimed at postgraduate students and early career postdocs providing in-depth tutorials encompassing concepts and software available to molecular microbiologists and microbial ecologists including:

Increasingly, natural product isolation strategies are circumventing culture-dependent methods for the isolation secondary metabolite genes directly from the environment. Many of these isolation strategies are undertaken with little knowledge of an environment's specific secondary metabolite potential. Next-generation sequencing technology was used to determine the diversity of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes within multiple environments to a depth previously not reported. A multiplexing strategy was used to amplify thousands of ketosynthase and amino acid condensation domain sequences from over thirty different environments. Sequences were differentiated according to function and taxonomic origin, as well as their distribution within distinct environments. Similar patterns of NRPS and PKS occurrence were observed between functionally similar but geographically distinct environments. Furthermore, increases in microbial diversity between environments did not influence the occurrence of these genes. It is expected that this approach will be applied to any environment enabling for the tailoring of culture-dependent and culture-independent strategies for the isolation of novel natural products.

In September, JAMS was back into top gear, with a bigger audience, and a room with a view. Kent Lim from Macquarie University led off with a talk on his PhD work on the biocontrol agent Pseudomonas strain Pf5. As is often the case in science, things didn’t work out as expected, and Kent found that knocking out suspected pyochelin transporters led to an increase rather than a decrease in efflux of this siderophore and its metabolic precursors. Kent valiantly soldiered on, applying qRT-PCR and Biolog phenotype microarrays to untangle the problem, but unfortunately, this released even more worms from the seemingly-bottomless can provided by strain Pf5. It seems that these transporters may in fact also be regulatory proteins, explaining the unexpected pleiotropic effects of the knockouts.